PICHIA, OPTIMIZATION OF PROTEIN
`EXPRESSION
`
`KOTI SREEKRISHNA
`Procter & Gamble Co.,
`Cincinnati, Ohio
`
`INTRODUCTION
`
`A wide variety of expression systems for production of pro-
`teins of interest have become available with the advent of
`recombinant DNA technology since the 1970s. The various
`expression systems include those based on bacteria, lower
`eukaryotes (yeasts and fungi), invertebrates (insect cells
`and larvae), vertebrates (cells and transgenic animals),
`and plants (cells and transgenic plants). Each of these
`systems has unique advantages. Yeasts and fungi have
`robust growth characteristics like bacteria. They are also
`readily amenable to genetic manipulation. Furthermore,
`they can perform many posttranslational modifications
`found in higher eukaryotes. The purpose of this article is
`to highlight the strategies that have been used for opti-
`mal protein expression with the Pichia yeast expression
`system, which in the past few years has turned out to be
`versatile and impressive yeast for production of proteins.
`
`BACKGROUND
`
`Pichia pastoris is a methylotrophic yeast. It is able to
`use methanol as sole carbon source for energy as well
`as for growth (1,2). It was originally developed by Phillips
`Petroleum Company (Bartlesville, Oklahoma) as an organ-
`ism of choice for bioconversion of natural gas (methane)
`into food (single cell protein). This may sound ironic,
`because nowadays every one is talking bioconversion of
`food crops to fuels (biofuels). Phillips Petroleum Company
`in the 1970s developed an efficient Pichia fermentation
`process [cell density >130 g dry cell weight per liter and
`biomass productivity >10 g/(L h)] (3,4). Though impres-
`sive, it could not compete with the economics of production
`of soy protein. After this set back, Phillips in the early
`1980s directed its future course with Pichia into two areas:
`
`1. Speciality Food or Feed Application: A rather impres-
`sive 100,000 L fermentation plant to churn out tons
`of Pichia for potential speciality food applications
`was completed in 1988. However, by 1993, the idea
`to use Pichia for speciality food/feed was abandoned
`as Pichia did not have generally regarded as safe
`(GRAS) status and Phillips Petroleum Company
`decided to focus on its core business of oil explo-
`ration, production, and petrochemicals.
`
`2. Development of Pichia Expression System for Pro-
`duction of High Value Recombinant Proteins: Pichia
`possesses a highly regulated pathway for the utiliza-
`tion of methanol (2,4). Synthesis of the enzymes
`of methanol metabolism are undetectable in the
`absence of methanol, but increase rapidly when
`methanol is used as the sole carbon source. The
`most dramatic effect is seen for alcohol oxidase
`(AOX1), which accounts for >30% of cellular protein
`(5). An extensive proliferation of the peroxisomes,
`known to sequester AOX1 and dihydroxy acetone
`synthase, is also observed in cells grown on methanol
`(5–7). Perhaps the decision of Phillips to exploit the
`above noted unique features of Pichia to develop
`a protein expression system was highly fortuitous,
`because the molecular genetic tools that had just
`become available for Saccharomyces cerevisiae, such
`as yeast–E.coli shuttle vectors, transformation pro-
`tocols, and site-specific integrative transformation
`(8,9) were found to be readily applicable to Pichia.
`With the advent of cloning of AOX1 promoter (10,11),
`availability of auxotrophic Pichia strain [GS115
`(his4) developed by George Sperl] and Pichia trans-
`formation protocols (12,13), successful high level
`expression of several proteins was readily demon-
`strated. They include, intracellular expression of
`Hepatitis B surface antigen particles (14), E. coli
`β-galactosidase (LACZ) (15), human tumor necro-
`sis factor (TNF) (16), as well as greater than gram
`per liter level of secretion of S. cervesiae invertase
`(SUC2) (17), and human serum albumin (HSA) (18).
`Pichia expression technology patent was granted to
`Phillips Petroleum Company in 1988 (11) and thus,
`by the late 1980s, Pichia expression system was well
`on its way to leave the nest.
`
`In the fall of 1988, Phillips Petroleum Company made a
`conscientious decision to license the system to other com-
`panies. Fortunately for Phillips licensing team (Katherine
`Bartosh, L.V. Benningfield, Mary Jane Hagenson, Jack
`Phillips, and Koti Sreekrishna), their very first commer-
`cial licensee (Glaxo–Wellcome) with just 2 days of training
`at Phillips produced some impressive expression results
`(19–23). Phillips was able to license the technology to
`20 companies in 4 years. Along side, it also started dis-
`tributing the Pichia expression kit, free of cost to any
`academic institution that approached the company for the
`kit. This was getting out of hand, and thus the company
`transferred distribution rights, free of cost, to Invitrogen
`Corporation (Carlsbad, California) in 1993. Since then,
`Invitrogen Corporation (http://www.invitrogen.com) has
`been aggressively distributing Pichia expression kit at
`reasonable cost and with some user friendly modifica-
`tions. They are largely responsible for wide usage of Pichia
`technology. In the same year (1993), Phillips sold Pichia
`technology to Research Technology Corporation (Tucson,
`
`Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. Flickinger
`Copyright © 2010 John Wiley & Sons, Inc.
`
`1
`
` 10.1002/9780470054581.eib480, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/9780470054581.eib480 by University of California - Los Ange, Wiley Online Library on [18/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Motif Exhibit 1021, Page 1 of 16
`
`Case No.: IPR2023-00322
`U.S. Patent No. 10,273,492
`
`

`

`2
`
`PICHIA, OPTIMIZATION OF PROTEIN EXPRESSION
`
`Arizona) (http://www.rctech.com). They have licensed the
`technology thus far to 160 companies. To date, >500 pro-
`teins have been expressed in the Pichia yeast (24) and
`over 3000 scientific articles published. There are over 100
`publications on fermentation optimization alone.
`Hepatitis B vaccine and interferon-α derived from
`P. pastoris have been commercialized in India since
`1999 and 2002, respectively, by Shantha Biotechnics
`(http://www.shanthabiotech.com) (25). Now the company
`makes >100 million units of hepatitis vaccine per year
`and sells these vaccines in 50 countries. The cost of
`vaccine has dropped to 15 cents a dose (24). Recombinant
`human insulin produced in Pichia is also marketed in
`India since 2003, by joint venture between Shantha
`Biotechnics and Biocon (http://www.biocon.com) (24,25).
`HSA expression technology originally demonstrated
`at Phillips Petroleum Company (18) and transferred to
`Green-Cross Corporation (Osaka, Japan) has been further
`developed and scaled up to produce one million dosage
`vials (12.5 g per vial) of authentic rHSA/year by Mitsubishi
`Pharma Corporation (Osaka, Japan) (26,27). This truly is
`a testimony to the high expression level and scale-up possi-
`ble with Pichia (25). (Please refer to http://www.rctech.com
`for a more complete list of Pichia-produced products under
`various stages of commercialization.)
`
`STRATEGIES FOR OPTIMIZATION OF PROTEIN
`EXPRESSION
`
`The typical Pichia expression vectors are all yeast–E. coli
`shuttle plasmids (see ‘‘Glossary of P. pastoris Vectors’’).
`The most commonly used vectors are based on AOX1 pro-
`moter (10,11) (see section titled ‘‘Alternative Promoters
`for Expression’’ below for other promoters available for
`Pichia expression). Numerous selectable marker genes
`available include HIS4 (His+ selection) (12), ARG4 (Arg+
`selection) (28), ADE1 (Ade+ selection) (29), and URA3
`and URA5 (Ura+ selection) (29,30), which can be used
`in conjunction with the appropriate Pichia auxotrophic
`strains. Several dominant selection markers that can be
`used for transformation of any P. pastoris strain are
`also available. These include SUC2 (allows growth on
`sucrose) (13), KanR (G418/Geneticin selection) (23,31),
`ZeoR (Zeocin selection) (32), BsdR (Blasticidin selection)
`(http://www.invitrogen.com), FLD1 (Formaldehyde selec-
`tion) (33), and SorR (Soraphen A selection) (34).
`A wide assortment of proteins have been produced in
`Pichia, which include human insulin (35), glucagon-like
`peptide (GLP) (36), G-protein coupled receptors (GPCRs)
`(37), Aquaporin (AQP1) (38), as well as correctly assem-
`bled human collagen fibers (39) and fibrinogen chains (40).
`These are among >500 proteins that have expressed in
`this system (24). Obviously, not all proteins are expressed
`at multiple grams per liter range. The expression level
`is largely influenced by inherent properties of a protein.
`Some proteins are readily expressed at high levels with
`minimal manipulation, while some proteins barely reach
`milligram levels, despite tremendous effort. Encourag-
`ingly, in many instances, the initial production yield of a
`protein can be dramatically enhanced by addressing the
`
`multiple factors that influence protein expression (41–43).
`The purpose of this review is to highlight the various
`expression-optimization strategies.
`
`Cellular State of the Expression Cassette
`The expression cassette can be introduced into Pichia by
`way of chromosomal integration or autonomous replica-
`tion. Chromosomal integration is preferable because of
`the following advantages: (i) stability of expression cas-
`sette; (ii) ability to generate clones that stably maintain
`multiple copies of the expression cassette (see section titled
`‘‘Gene Dosage: Exploiting the Clonal Variation of Expres-
`sion’’); (iii) control over the site of integration (for example,
`AOX1, HIS4, ARG4, URA3 loci); and (d) ability to engineer
`different modes of integration (with or without eviction of
`the AOX1 coding sequences) (see section titled ‘‘Methanol
`Utilization Phenotype of the Host: Mut+ or Muts’’).
`Plasmids based on autonomous replication sequence
`(ARS) such as pHIL-A1 (see ‘‘Glossary of P. pastoris Vec-
`tors’’) although can be introduced into Pichia cells with a
`high transformation frequency, they are rapidly lost from
`the population of dividing cells, and eventually integrate
`at one or more of the homologous sites on the chromo-
`some (12,13). Owing to their ability of transform Pichia at
`high frequency (>105 μg−1) and ease of plasmid rescue,
`the autonomous plasmids are useful for cloning genes in
`Pichia by functional complementation.
`
`Site of Integration of the Expression Cassette
`The AOX1 promoter used in Pichia expression vectors is
`active irrespective of the site of integration (AOX1, HIS4,
`ARG4, ADE1, or URA3 loci). However, AOX1 locus is the
`preferred site of integration for stable expression, because
`integration at the other loci can result in loss of the expres-
`sion cassette due to intrachromosmal cross over between
`the mutant and good copy of the gene (unpublished obser-
`vations). If the vector also contains a dominant selection
`marker (discussed earlier), then selection pressure can be
`applied for stable maintenance of expression cassette at
`those sites.
`
`+
`
`or Muts
`Methanol Utilization Phenotype of the Host: Mut
`Transformation of a P. pastoris his4 strain (GS115) using
`linear DNA expression cassette with the ends bearing
`homology to the 5(cid:2) and 3(cid:2) regions of the AOX1 chro-
`mosomal locus results in the site-specific eviction of the
`AOX1 structural gene at a high frequency (5–20% of the
`His+ transformants) (16,28). Such clones can be readily
`distinguished by replica-plating the colonies from the ini-
`tial His+ selection plate on to a minimal methanol (MM)
`medium plate. The clones that have undergone eviction of
`AOX1 grow slower (MutS) compared to Mut+ clones with
`an intact AOX1 gene, because, in such clones, growth on
`methanol is dependent on the alcohol oxidase encoded by
`AOX2, which is expressed at much lower level (weaker
`promoter) (44). The Mut+ clones arise due to circulariza-
`tion of the linear DNA expression cassette inside the yeast
`cell prior to integration. Thus, both MutS and Mut+ clones
`result in the same experiment.
`
` 10.1002/9780470054581.eib480, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/9780470054581.eib480 by University of California - Los Ange, Wiley Online Library on [18/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Motif Exhibit 1021, Page 2 of 16
`
`Case No.: IPR2023-00322
`U.S. Patent No. 10,273,492
`
`

`

`Alternatively, one can use the Pichia strain KM71 (his4
`arg4 aox1 :SARG), which has been rendered MutS by
`replacing much of the chromosomal AOX1 with S. cere-
`visiae ARG4 (28).
`For intracellular expression, it is critical to use MutS
`cells because they will have a lower level of AOX1 and the
`expressed protein of interest can be more readily purified.
`Furthermore, the precious cellular machinery can be more
`fully utilized for the expression of protein of interest.
`For protein secretion, the choice of Mut+ or MutS is less
`stringent and more a matter of preference as the impact is
`more on feed protocols than the secretion level. In fact, the
`secretion levels of HSA are similar with both Mut+ and
`MutS cells (45).
`
`Gene Dosage: Exploiting the Clonal Variation of Expression
`Early on a dogma prevailed among the researchers of
`the Pichia system, based on limited experience (14,15),
`that increasing the gene dosage did not impact expression
`because of the remarkable strength of the AOX1 promoter.
`This dogma was shattered on the Good Friday of March
`28, 1986, with my observation of dramatic clonal vari-
`ation (<1% to >30%) in the expression of human TNF,
`that was subsequently attributed to gene dosage. Fur-
`thermore, the high copy number was stably maintained
`even after several days of growth in high cell density fer-
`mentor. The observation with TNF expression was readily
`exploited for expression of tetanus toxin fragment C (21),
`Bordetella pertussus pertactin P69 (20), and mouse epi-
`dermal growth factor (EGF) (19), and has been a key
`strategy for successful expression of hundreds of proteins
`in the Pichia system. However, in numerous cases, a single
`copy of the expression cassette is sufficient and deliber-
`ately increasing the copy number had no significant effect
`on the production (14,15,17,18). In some rare instances,
`an increase in copy number has a deleterious effect on
`the production level as has been noted with secretion of
`human insulin-like growth factor (IGF) (46) and Necator
`americanus (hookworm) secretory protein (Na-ASP1) (47).
`Because the effect of gene copy number on expression
`is unpredictable, it is prudent to examine the production
`level as a function of gene dosage. The spheroplast method
`of transformation of Pichia results in transformants with
`a wide range of copy numbers (16,48). Evaluation of as
`few as 100 individual clones for protein production is
`generally adequate to arrive at a good producer. Though
`bit laborious, spheroplast method of transformation yields
`clones with wide range of gene dosage at a high fre-
`quency (48). If other methods of transformation such as
`using LiCl or electroporation, which do not yield high
`frequency of multicopy clones, then one can use more effi-
`cient screens. These include use of colony hybridization
`with DNA probes or using appropriate vectors that would
`allow selection based on increased level of resistance to
`one of G418/geneticin (23,31), zeocin (32), formaldehyde
`(33), blasticidin (http://www.invitrogen.com), or soraphen
`A (34). Increased drug resistance does not automatically
`ensure multicopy integration and several dozen resistant
`colonies must be analyzed for copy number and expression.
`Another approach to identify high producers may be
`to use a visual tag to the protein being expressed. In one
`
`PICHIA, OPTIMIZATION OF PROTEIN EXPRESSION
`
`3
`
`instance, C-terminal green fluorescent protein fusion was
`used as a visual reporter to identify human Mu-opioid
`receptor expressing clones (49). Rhizopus oryzae lipase
`(ROL) bearing an N-terminal GFP tag was more efficiently
`secreted in Pichia compared to one with a C-terminal
`GFP tag (50). Recently, a fusion protein ZZ-EGFP con-
`sisting of ZZ domain of staphylococcal protein A (SpA)
`with enhanced green fluorescent protein (EGFP) was also
`secreted in the Pichia, with a hexahistidine tag (51). GFP
`tag may serve as a visual reporter to readily identify high
`producing clones. It can also be useful for on-line monitor-
`ing of protein production in the fermentor (52) However,
`on-line monitoring can be complicated by high levels of
`riboflavin (excitation/emission = 450/530 nm) secreted by
`Pichia (50), which in itself has been used for monitoring
`biomass production (53). By using a fluorescent protein
`tag outside the interfering range, one may be able to reli-
`ably monitor simultaneously both cell and product yield
`on-line.
`Vectors such as pAO815 (54,55) have also been
`described that would allow in vitro construction of
`expression cassette concatamers. This approach is useful
`to accurately correlate copy number to expression level
`over a narrow range of gene dosage.
`(cid:2)
`Untranslated Region
`Translational Optimization: 5
`The nucleotide sequence and the length of the 5(cid:2) untrans-
`lated region (5(cid:2)UTR) are detrimental to optimal protein
`translation. The leader length of the highly expressed
`AOX1 mRNA is 114 nucleotides long, and the sequence
`is A + U rich (10,11). For optimal synthesis of heterolo-
`gous proteins, it is essential that the 5(cid:2)UTR should closely
`resemble that of the AOX1 mRNA. Ideally, it is preferable
`to make it identical to that of AOX1 mRNA. The expres-
`sion level of HSA is increased >50 fold by optimizing the
`5(cid:2)UTR to mimic that of AOX1 mRNA (18). Expression
`plasmids such as pHIL-D7 (42) can be used to make an
`exact construct. This plasmid has unique Asu II and Eco
`RI sites immediately following 5(cid:2)AOX1. The second Asu
`II site that was originally present in 3(cid:2)AOX1 has been
`eliminated. Therefore, the sequence TTCGAAACG can be
`added immediately upstream of the ATG start codon of
`the gene of interest, and an Eco R I site can be engineered
`downstream of the stop codon for insertion at Asu II–Eco
`RI sites of pHIL-D7 (42).
`
`Transcriptional Optimization
`Genes with high A + T nucleotide clusters are poorly
`transcribed in Pichia due to premature termination of the
`transcription. For example, ATTATTTTATAAA, present
`in HIV-gp120 has been identified to block transcription
`in P. pastoris, and the premature termination is over-
`come by altering the sequence to TTTCTTCTACAAG (22).
`Because we are not aware of all the problematic A + T rich
`clusters, a general strategy with A + T rich genes is to
`redesign them using P. pastoris preferred codons (48,54),
`http://www.kazusa.or.jp/codon/) so as to have an A + T
`content in the range of 30–55%. By using this approach, it
`has been possible to construct Pichia strains for efficient
`production of several proteins, which include tetanus toxin
`
` 10.1002/9780470054581.eib480, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/9780470054581.eib480 by University of California - Los Ange, Wiley Online Library on [18/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Motif Exhibit 1021, Page 3 of 16
`
`Case No.: IPR2023-00322
`U.S. Patent No. 10,273,492
`
`

`

`4
`
`PICHIA, OPTIMIZATION OF PROTEIN EXPRESSION
`
`fragment C (20), Bacillus sphaericus BSP1 and BSP2 het-
`erodimeric mosquitocidal toxin (55), anticoagulant protein
`ghilanten (56), spider silk proteins (57), human endostatin
`(58), and human lactoferrin (59).
`
`Product Secretion
`For a protein which is normally secreted, that is the pre-
`ferred mode of expression, for multiple reasons. It is easier
`to recover product from extracellular medium as there will
`be relatively fewer proteins. Also, many proteins that are
`normally secreted remain predominantly insoluble when
`expressed in the intracellular compartment, as has been
`seen with HSA and salmon growth hormone (unpublished
`observations). Likewise, a protein that is not normally
`secreted may be difficult to secrete. However, in some
`cases, such as human TNF, it is possible to express at high
`levels in the intracellular compartment (16). Interestingly,
`the intracellular human tetrameric catalase has success-
`fully been expressed as a secreted protein in Pichia (60).
`A wide variety of heterologous proteins have been
`secreted in Pichia. In several instances, HSA (18), inver-
`tase (17), bovine lysozyme (61), barley alpha amylases
`(62), cathepsin E (63), and thaumatin (64) the native
`signal sequence is adequate. In the case of matrix met-
`alloproteinases, although native signal sequences work
`(41), both secretion and product yield are improved while
`using the S. cerevisiae pre-proalpha mating factor (αMF)
`secretion signal sequence (65,66). Likewise, with Candida
`rugosa lipase 1, although native signal works, both prod-
`uct stability and yield are tremendously improved with the
`use of pre-proαMF signal sequence (67). For thaumatin
`secretion, native signal works, but not the pre-proαMF
`signal sequence (64), whereas for human interferon-alpha
`2b (IFN-a2b), native signal sequence does not work, but
`pre-proαMF works (68).
`Pre-proαMF secretion signal sequence has worked
`well for secretion of large variety of proteins, including
`the smaller-sized products such as aprotinin (69), EGF
`(19,69), IGF-1 (70), and ghilanten (56). The processing of
`pre-proαMF secretion signal involves three steps. The first
`is the elimination of the pre-region by signal peptidase,
`second, KEX2, and YPS proteinases cleave out the prore-
`gion (71,72), and finally, N-terminal Glu–Ala repeats
`are removed by the action of dipeptidyl-aminopeptidase
`(Dpap). The efficiency of each processing step depends on
`amino acid sequence adjacent to the processing site as
`well as the tertiary structure of the secreted protein. All
`these factors contribute to incomplete processing and/or
`reduced yield of mature protein (73–75).
`it is
`In making αMF signal sequence constructs,
`generally preferable to retain the Glu-Ala spacers
`adjacent to the Kex2-like protease cleavage site ( . . .
`Val-Ser-Ser-Leu-Glu-Lys-Arg-Kex2pGlu-Ala-Dpap-Glu-Ala-
`Dpap-fused protein). The presence of Glu-Ala spacers
`help to alleviate the steric interference imposed by
`the fused protein, resulting in an efficient cleavage of
`the prosequence by the Pichia Kex2 like protease (69).
`The Glu-Ala spacer is subsequently cleaved by diamino
`peptidase (Dpap coded by STE13) to yield the protein
`of
`interest free of additional N-terminal amino acid
`
`residues. Interestingly, it has recently been reported that
`the pre-proαMF without the Glu-Ala repeats directed the
`secretion of correctly processed human IFN-a2b at 200
`mg/L level, whereas the pre-proαMF having the Glu–Ala
`spacer, although secreted an equivalent amount of
`IFN-a2b, the secretion signal was inefficiently processed
`(68). Thus, ideally all variations must be explored to
`arrive at the best fit for a given protein.
`Other secretion signal sequences used in Pichia include
`those of acid phosphatase (Pho1p), hybrid of Pho1p secre-
`tion signal with αMF and Kex2p cleavage site, invertase,
`lysozyme, HSA, Phaseolus vulgaris agglutinin, inulinase,
`and alpha amylase, glucoamylase, and S. cerevisiae killer
`toxin I (42,59,76).
`
`Choice of the Secretion Signal
`Only in a limited number of cases has a thorough com-
`parison been made on the relative efficacies of different
`signal sequences and variations thereof (for example,
`(59,64,68,69)). In the case of invertase secretion, both the
`extent of glycosylation and secretion rate are enhanced
`when the native signal sequence is substituted with the
`pre-proαMF signal sequence (69), although both yields are
`greater than gram per liter level of the product. However,
`in the case of proteins that are more susceptible to pro-
`teolysis, improvement in secretion rate can increase pro-
`duction level as has been noticed with MMP-1 (41,65,66).
`Based on the relatively high success of pre-proαMF signal
`sequence, it makes sense to test that signal (and variations
`there of) first, side by side with the native secretion signal
`sequence, before exploring other signal sequences.
`
`Production Enhancement by Manipulation of Media and
`Growth Conditions
`More often than not, a secreted protein is rapidly degraded
`in the Pichia medium due to extracellular proteases,
`cell-bound proteases, as well as by intracellular pro-
`teases released to medium due to cell death and cell lysis.
`Multiple stress factors (especially in high cell density fer-
`mentor), which include starvation, pH shift, temperature
`change, change of carbon source, buildup of toxins, and
`reactive oxygen species (ROS) are considered to cause cell
`death and cell lysis (77).
`One of the approaches to increase the stability and
`yield of the secreted protein in the culture medium is
`by manipulating the pH of the medium to arrive at the
`optimal pH for blocking a problem protease. Suggested
`pH range for experimentation is between 2.8 and 8 (see
`section titled ‘‘Media Compositions’’). This pH range does
`not affect the growth significantly. HSA yield was sig-
`nificantly improved by raising the pH from 5.2 to 6,
`with adequate aeration. The yield was further enhanced
`by the addition of yeast extract (1%) and peptone (2%)
`(45,48,78,79). Production of mouse EGF was favored at pH
`6 in the presence of casamino acids (19). Casamino acids is
`preferable to yeast extract + peptone, because the peptide
`components of peptone (such as collagen fragments) can
`interfere in product analysis and recovery (41). For both
`IGF-I (70) and cytokine growth-blocking peptide (80), pH
`3 was found to be optimal. Greater than twofold increase
`
` 10.1002/9780470054581.eib480, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/9780470054581.eib480 by University of California - Los Ange, Wiley Online Library on [18/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Motif Exhibit 1021, Page 4 of 16
`
`Case No.: IPR2023-00322
`U.S. Patent No. 10,273,492
`
`

`

`in production of cellulose-binding module cellulose 6A and
`lipase B (CBM-CALB) is noted by reducing the pH from 5
`to 4 in a bioreactor run (81).
`Hirudin variant 2 (rHV2) secreted in Pichia was found
`to be degraded to four active fractions, including the intact
`rHV2 (Hir 65) and three C-terminally truncated forms
`Hir 64, Hir 63, and Hir 62 (82). Degradation of hirudin
`was reduced by using growth-rate-limiting quantities of
`methanol (3.09 g/L methanol; specific growth rate main-
`tained at 0.02–0.047 h–1) (82). Degradation of hirudin
`+ to relieve the stress
`was also reduced by increasing NH4
`of nitrogen starvation (83). Ascorbic acid (4 mmol/L) was
`used to quell ROS and improve cell viability. Under these
`conditions intact and total hirudin production reached 2.90
`and 5.03 g/L, respectively, in contrast to 1.75 and 4.70 g/L
`in the absence of ascorbic acid (84).
`Lower cultivation temperature is shown to improve
`product yield in several
`instances, which include
`◦
`◦
`CBM-CABL (22
`C) (81), laccase (20
`C) (85), galactose
`◦
`◦
`oxidase (25
`C) (86), Rh midkine (20
`C) (87), human AFP
`◦
`◦
`C) (88), herring proteins (23
`C) (89), and human
`(23
`◦
`bile salt activated lipase (BSSL, 20
`C)
`(90). Pichia
`◦
`C, greatly
`cultivated at temperatures as low as 15
`enhanced the yield of ScFV production due to reduced
`protease level (91). Secretion level of bivalent T cell
`immunotoxin A-dmDT390-bisFy(G4S) was also enhanced
`◦
`at 15
`C, which was attributed to reduced protease levels,
`improved folding, as well as reduced toxicity of the
`immunotoxin to Pichia (92). Thus, we may say that the
`low temperature expression helps to increase the yield of
`aggregation-prone, unstable, and toxic products in Pichia.
`Addition of 5 mM EDTA to the medium also improves
`accumulation of proteins expressed in Pichia (41).
`Supplementation of the induction medium with 0.4 M
`L-arginine, 5 mM EDTA, or 2% casamino acids in the
`BMMY induction medium (see section titled ‘‘Media
`Compositions’’) increased scFV expression (91). It should
`be noted that medium manipulation can significantly
`alter the profile of endogenous protein components
`in the culture media, such that previously unnoticed
`proteins accumulate at high levels. For example, we
`have noticed that addition of 5 mM EDTA causes
`accumulation of a protein of approximately 50 kDa
`in the extracellular medium (41). This protein has
`the
`aa
`sequence DIIWDYSSEKIMGVNLGGWL. . .,
`which matches closely with the exo-β-1,3-glucanase of
`S. cerevisiae (93) and Candida albicans (94). We have
`also noticed that overexpression of human tissue inhibitor
`of matrix metalloproteinase (TIMP1) in Pichia leads to
`greater gram per liter accumulation of a 18–22 kDa
`Pichia protein of sequence ADYMC?GLAIYGAWEC?
`GPEAGPFDSEC?LLATD (41).
`
`Production Enhancement Using Protease-Deficient Strains
`In addition to the optimization of media and growth condi-
`tions, the product yield can be further improved by using
`a protease-deficient Pichia strains generated by knock-
`ing out a Pichia protease or by overexpressing a protease
`inhibitor (for example, TIMP1 strain as noted above).
`Protease-deficient Pichia strains SMD1168 (his4, pep4),
`SMD1165 (his4, prb1), and SMD1163 (his4, pep4, prb1)
`
`PICHIA, OPTIMIZATION OF PROTEIN EXPRESSION
`
`5
`
`have been constructed (95). These strains have a disrup-
`tion in the genes encoding proteinase A (PEP4) and/or
`proteinase B (PRB1) (56). Proteinase A is a vacuolar
`aspartyl protease necessary for the activation of vacuo-
`lar proteases, such as carboxy peptidase Y and proteinase
`B. In the absence of proteinase A, Prb1p is not fully
`active. Thus, pep4 strain (SMD1168) is also expected to
`have a lower level of Proteinase B activity. The prb1
`strain (SMD1165) lacks proteinase B, whereas pep4prb1
`strain (SMD1163) lacks all three of the protease activities.
`These strains in general have low storage viability and are
`less robust (42,43). Nevertheless, these protease-deficient
`strains, in combination with other strategies to improve
`product stability, have been used to improve the pro-
`duction yield of IGF-1 (46), ghilanten (56), laccase (96),
`and human catalase (60). Disruption of KEX1, which
`codes for a carboxy peptidase, resulted in expression of
`full-length endostatin in Pichia (97). Recently, using a
`gene pop-in/pop-out gene replacement approach (98), the
`KEX1 was deleted from a Pichia hirudin production strain.
`This resulted in most significant improvement in intact
`Hir65 production, which approached 2.4 g/L for the KEX1
`deleted strain compared to 1.1 g/L seen with the strain
`without KEX1 deletion (99).
`
`Enhancement of Protein Secretion by Overexpression of
`Chaperone Proteins
`Pichia is able to perform many posttranslational modifi-
`cations found in higher eukaryotes, which include correct
`folding, disulfide bond formation. Folding and disulfide
`bond formation in some cases can be the rate-limiting step
`in protein expression leading to protein aggregation (100).
`It is not clear which single chaperone is most important or
`which combination optimally cooperates in this process.
`Overexpression of Pichia protein disulfide isomerase
`(PDI), which is important for protein folding in the endo-
`plasmic reticulum (ER), was able to increase the secretion
`of Na-ASP1 protein in high copy clones (47). As noted
`before, high copy clones of Na-ASP1 secreted less material
`than single-copy constructs, perhaps due to overburdening
`the Pichia secretory/protein folding machinery, which was
`corrected by PDI overexpression.
`A33scFV in Pichia is expressed at 4 g/L level, which
`rose to >10 g/L by overexpression of immunoglobulin bind-
`ing protein (Bip) (101). The noted impressive increase is
`attributed to increase in folding capacity. PID overex-
`pression did not have any effect on A33scFV expression.
`This was unexpected because A33scFV contains disulfide
`bonds. Furthermore, simultaneous overexpression of both
`BiP and PDF also did not have any effect in this system. It
`was also noted that PDI expression in the A33scFV strain
`caused a six-fold increase in endogenous BiP expression,
`suggesting that PDI was inducing an unfolded protein
`response due to excess chaperone and recombinant pro-
`tein in the ER. In another study it was found that the
`chaperone combinations YDJiP/PDI, YDJiP/Sec63, and
`Kar2p/PDI synergistically increase secretion levels 8.7,
`7.6